The trabecular meshwork of the eye is the principal site of outflow resistance to the aqueous humor. Our long-term goal is to develop selective gene transfer systems to assist cells in reducing resistance to aqueous humor outflow and thereby stopping visual field loss. We looked at the effects of transforming growth factor beta (TGF- beta) on the trabecular meshwork since the level of this cytokine increases in the aqueous humor with glaucoma. Using gene array analysis we were able to see alterations in gene expression of components related to the extracellular matrix when (TGF-beta) was added to the culture media of human trabecular meshwork cells. We verified the results of the gene arrays with real time polymerase chain reactions and with relative quantitative polymerase chain reactions. In addition, analysis of the proteome of the cells treated with TGF-beta was done. Several of the genes that were altered as a result of the TGF treatment had changes in the protein content within the cells. In addition, we noted changes in several cytoskeletal proteins. These changes suggested that in addition to the extracellular components whose gene expression had changed as a result of the TGF-beta treatment, alteration in the cytoskeleton of the trabecular meshwork was influenced at the protein level. Both of these finding are consistent with work that has been published with trabecular meshwork in perfusion culture and are consistent with changes observed in glaucoma. Thus, several of the alteration in the trabecular meshwork thought to be related to glaucoma may in fact be related to the increase in TGF-beta in aqueous humor. We are also studying the protein myocilin. Mutations in this protein have been shown to be associated with some forms of glaucoma. We over-expressed normal myocilin in mouse eyes to determine if the intraocular pressure was altered. Although the amount of myocilin in the mouse eye was approximately 4.7 fold more than in human aqueous humor, or about 2ng/ul, no increase in intraocular pressure was observed. These results indicate that over-expression of human myocilin by itself is not the cause of the increased resistance to outflow seen in primary open angle glaucoma. In addition, we expressed mutated myocilin to determine how this protein might be altering trabecular meshwork cells and thereby causing glaucoma. In vivo, in our transgenic mice, the mutated myocilin remained in the endoplasmic reticulum and the build-up of this mutated protein is probably responsible for the alteration and death of the trabecular meshwork cells in this form of glaucoma. We are currently working on ways to silence mutated myocilin. Silencing this protein should ultimately help the cells in regaining normal function and thereby cause an increase in outflow. This would have the effect of reducing the damage to the retinal ganglion cells.